The production of high quality isotope-enriched protein samples is a prerequisite for applying modem NMR methods for protein structure determination. Although NMR spectroscopy is well suited for rapid semi-automated structure determination of small proteins (MW<15 kDa), solving structures of larger proteins and multimeric complexes is considerably more challenging. As the size of the molecule increases, so does the molecule's rotational correlation time and, consequently, the efficiency of 1H—1H relaxation mechanisms. One way to suppress these effects is to incorporate deuterium into the protein sample, diluting the 1H—1H relaxation networks and increasing 13C and 15N relaxation times, resulting in sharper line widths for 13C, 15N and remaining 1H nuclei, and dramatically improved signal-to-noise ratios. Perdeuteration is generally required for studies of larger proteins, particularly membrane proteins. 2II,13C,15N-enriched protein samples are also central to certain strategies for fully automated analysis of small protein structures.
While deuterium incorporation into protein samples can greatly improve the quality of data collected, the sample preparation itself can be challenging. Cell growth is affected by increasing 2H2O concentration, and cells must be gradually acclimated to high 2H2O concentration in incremental steps. Once acclimated, additional isotopically-enriched reagents are introduced, and protein expression can proceed. However, the overall protein yield in these conditions is often significantly reduced. As fermentation media costs for production of uniformly 2H,13C,15N-enriched samples range from $1500-$3,000 per liter, or higher, perdeuteration methods are generally employed only when absolutely required; for this reason, many potential applications of perdeuterated samples in routine protein NMR applications have not been pursued.
Additionally, expression of the target gene prior to culture condensation and resuspension in isotope enriched medium leads to heterogeneously labeled protein and results in the accumulation of approximately 10-20% of the total protein completely unlabeled.
Thus there remains a need for a process that is more cost effective, and results in a higher percentage of labeled protein.